The metastatic spread of epithelial cancer cells from the primary tumor to distant organs necessitates tumor cells to gain mesenchymal characteristics and to lose epithelial features, a phenomenon known as the epithelial-mesenchymal transition (EMT). Various factors, including cytokines/growth factors in tumor microenvironment, transcription factors belonging to homeobox (HOX) super-family and microRNAs (miRNAs), can induce EMT. In a systematic evaluation of miRNA:mRNA pair important for breast cancer cell EMT, we found that miR-375 is present in high abundance in epithelial-like cells but very low in mesenchymal-like ones. Among miR-375's putative targets, SHOX2 is expressed at high level in mesenchymal-like breast cancer cells but lowly expressed in epithelial-like ones. To investigate the role of miR-375:SHOX2 pair in breast cancer cell EMT, we found that enforced miR-375 expression is sufficient to abrogate EMT traits in mesenchymal-like breast cancer cells while SHOX2 overexpression can potently induce EMT in epithelial-like cells. We discovered a double negative miR-375/SHOX2 feedback loop in which miR-375 suppresses endogenous SHOX2 expression by directly targeting SHOX2's 3'-UTR whereas SHOX2 represses miR-375 transcription through the action of Lysine (K)-Specific Demethylase 5B (KDM5B). In addition, SHOX2 also promotes TGF receptor I (TRI) transcription, an event also capable of promoting EMT. Based on these findings, we formed our central hypothesis: miR-375/SHOX2 pair is dynamically involved in breast cancer cell EMT. Because of the well-recognized importance of EMT in breast malignancies, our goal of this proposal is to elucidate the basis of miR-375/SHOX2 feedback regulatory loop and the mechanisms underlying the dual action of SHOX2 in EMT induction. Moreover, we also intend to develop a therapeutic strategy through targeting events critical for SHOX2-induced EMT. Three specific aims are proposed in this application: 1) Understand how SHOX2 suppresses miR-375 expression in breast cancer cells; 2) Define mechanisms underlying SHOX2/miR-375 regulation of EMT in breast cancer cells; and 3) Determine the role of SHOX2 in breast tumorigenesis. The success of this application will have important impact in providing knowledge on both understanding EMT and building foundation for novel anti-cancer therapeutic modality.